The present invention relates to a novel enzyme capable of specifically hydrolyzing D-.alpha.-alanineamide to produce D-.alpha.-alanine (hereinafter referred to as D-amidase) and a process for producing D-.alpha.-alanine and/or L-o-alanineamide using the enzyme.
D-.alpha.-alanine is an important compound as a sweetening, or a synthetic intermediate or starting compound for synthesizing various physiologically active substances. L-.alpha.-Alanineamide is a starting compound for producing L-.alpha.-alanine, which is an important amino acid as food and medicines.
The following processes for producing D-.alpha.-alanine have been known so far.
(1) A microbiological process for producing D-.alpha.-alanine through direct fermentation [Japanese Published Unexamined Patent Application Nos. 22881/76 and 76482/77].
(2) A process for producing D-.alpha.-alanine by separating D-.alpha.-alanine from DL-.alpha.-alanine through the action of microorganism having an ability to decompose only L-.alpha.-alanine [Ohshima and Tanaka: Amino Acid Nucleic Acid, 15, 89-93 (1966)].
(3) A process for producing D-.alpha.-alanine through the action of acylase produced by a microorganism, upon the N-acyl compound of DL-.alpha.-alanine and through the successive optical resolution of DL-.alpha.-alanine (Japanese Published Examined Patent Application No. 22380/66).
(4) A process for producing D-.alpha.-alanine through the action of microorganisms having a hydantoinase activity upon 5-methylhydantoin to form D-(N-carbamoyl)alanine, and by treating D-(N-carbamoyl)alanine chemically or microbiologically [Yamada, et al: Hakko To Kogyo, 38, 937 (1980), Japanese Published Unexamined Patent Application Nos. 1189/78, 89088/79,88697/80, 104890/80, 114291/80, etc.].
(5) A process for producing D-.alpha.-alanine by hydrolyzing D-.alpha.-alanineamide through the hydrolytic activity possessed by microorganisms belonging to the genus Bacillus, Bacterium, Micrococcus, Brevibacterium, Achromobacter, Alcaligenes, Kurthia, Pseudomonas, Rhodococcus or Serratia [Japanese Published National Publication No. 500319/81,[Japanese Published Unexamined Patent Application Nos. 184392/85, 96989/86 and 274690/86].
(6) A process for producing D-.alpha.-amino acid by hydrolyzing DL-.alpha.-amino acid amide through the hydrolytic activity specific to D-.alpha.-amino acid amide and possessed by microorganisms belonging to the genus Rhodococcus [Japanese Published Unexamined Patent Application No. 87998/88].
(7) A process for producing D-.alpha.-alanine through the action of D-amino-acid transaminase upon pyruvic acid [Japanese Published Unexamined Patent Application No. 205790/87].
(8) A process for producing D-.alpha.-alanine by the chemical optical resolution through the preferential crystallization of DL-.alpha.-alanine p-chlorobenzenesulfonate [Japanese Published Examined Patent Application No. 14369/72 and Japanese Published Unexamined Patent Application No. 57914/73].
Among the aforementioned processes for producing D-.alpha.-alanine, the processes (1), (2), (6) and (8) have no or lower D-.alpha.-alanine productivity, and the processes (3), (4) and (7) have complicated operations because of the reaction consisting of several stages. The processes (5) and (7) require an expensive optically active substrate and thus raise the cost of production.
The enzymes capable of hydrolyzing D-.alpha.-alanineamide are disclosed in Abstracts of 1988th Meeting of the Agricultural Chemical Society of Japan, page 352.
Any process for producing L-.alpha.-alanineamide of high optical purity at industrially low cost is unknown yet.
Presently, an enzyme capable of producing D-.alpha.-alanine and L-.alpha.-alanineamide of high optical purity directly from inexpensive DL-.alpha.-alanineamide and a process for producing D-.alpha.-alanine and L-.alpha.-alanineamide using the enzyme are desired.
Extensive studies have been made on the development of an industrially favorable process for producing D-.alpha.-alanine from DL-.alpha.-alanineamide. As a result, the present inventors have found that microorganisms belonging to the genus Arthrobacter produce an enzyme capable of specifically hydrolyzing D-.alpha.-alanineamide thereby to produce D-.alpha.-alanine from DL-.alpha.-alanineamide or D-.alpha.-alanineamide. Examination of its physicochemical properties after isolation and purification of the enzyme reveals that the enzyme is novel.